1. Field of the Invention
The present invention is in the fields of biotechnology and food/beverage manufacturing. The invention relates to the production of malt beverages, and more particularly to the production of malt beverages having improved flavor-stability. In particular, the invention relates to methods and compositions for improving the flavor stability of fermented malt beverages such as beer, and to malt beverages produced by these methods.
2. Related Art
The Brewing Process
Overview. In the production of fermented malt beverages such as beer, a warm water extract of barley malt, with or without other unmalted grains such as rice or corn, is boiled with hops, cooled and then subjected to the fermentative action of yeast. The warm water used to extract the malt allows the action of several enzymes in the malt to hydrolyze the starch in the barley (and in the corn or rice) to fermentable sugar, which is acted on by the yeast to produce the alcohol in the fermented malt beverage.
Malting. Barley malt is steeped with water to produce steeped out barley which is germinated at a fairly low temperature. Germination is carried out with daily mixing and water addition as needed to maintain the moisture content at about 43%. The resulting green malt contains a high content of beer flavor precursors, beer flavor components, and coloring agents. After germination is complete, the green malt is heated at a high moisture content to generate beer flavor precursors, beer flavor components and also to reduce amylolytic enzyme activity. After heating, the malt is dried to a moisture content of 3.5-5.5% and a soluble protein content of 6.5-8%. The dried malt can then be mashed to produce a wort which is boiled with hops, cooled, pitched with brewers yeast, and processed by conventional brewing processes and in conventional brewing equipment.
Mashing. The malt, which may actually be a blend of malts (i.e., standard brewer""s malt, high color, low amylase malt, etc.), is ground and mixed with 2.5 to 4 times its weight of warm water in large tubs and mashed at 35-40xc2x0 C. for 5 to 15 minutes until it forms a thick malt mash. The mash is then permitted to rest for 45-90 minutes without stirring, then heated in steps to 70-73xc2x0 C. while stirring, with time allowed at each step for the various enzymes to convert the starches into fermentable sugars. Following heating, the mash is held for 15-30 minutes, the temperature is raised to 75xc2x0 C., and the mash is transferred to the lauter unit.
If rice and corn adjuncts are to be used, they are separately cooked and a cooker mash is obtained. Production of the cooker mash involves the use of adjuncts along with a 10%-30% portion of the malt (or the addition of commercial enzymes) in order to convert raw starch into fermentable sugars. The adjuncts and the malt portion are gradually brought to boiling and held there until the products are completely gelatinized. During the final stages of mashing (at the higher temperatures), the cooker mash and the malt mash are combined.
Mashing serves a three-fold purpose. First, it brings into solution those substances of malt (and adjuncts) which are readily soluble in warm water. Second, it permits malt enzymes to act on insoluble substances and render them soluble. Third, it provides a far-reaching enzymatic degradation of starches, proteins and gums into products of smaller size and lower molecular weight.
Lautering and Sparging. Lautering consists of the removal of the liquid, now termed the xe2x80x9cwort,xe2x80x9d from the insoluble husks or xe2x80x9cspent grains.xe2x80x9d Lautering begins upon termination of the mashing process, whereby the finished mash is transferred to a lautering tub. There it is allowed to rest for about ten to thirty minutes during which time the spent grains settle to the bottom. The lautering tub is equipped with a false bottom containing numerous perforations and an outlet leading to the true bottom of the tub. The mash is then allowed to settle for 10-20 minutes and run-off begun. The wort is recycled until reasonably clear. The clear wort is then pumped into a brewing kettle. Hot water is run through the spent grains to rinse out, or sparge, any remaining wort.
The lauter temperature is about 72-77xc2x0 C. for both the bath and sparge water. The amount of sparge water used is about 50-75% of the amount of brewing water.
Boiling and Hopping of Wort: Primary Fermentation. The wort is boiled vigorously for one to two and one-half hours in the brew kettle. Hops (or extracts thereof) may be added at various stages of the boiling process, depending on the nature of the final product that is sought.
Wort boiling serves a number of objectives, including (1) concentration of the sparged wort, (2) complete inactivation of enzymes that may have survived the final mashing process, (3) coagulation and precipitation of high-molecular weight proteins and solids (termed xe2x80x9ckettle breakxe2x80x9d or xe2x80x9chot breakxe2x80x9d), (4) extraction of desirable hop constituents, and (5) sterilization of the wort.
Cooling, Fermentation and Storage: Maturation. After boiling, the wort is strained to remove the solids, or xe2x80x9ctrub,xe2x80x9d and the wort is then cooled to a temperature of about 12-16xc2x0 C.
Fermentation is initiated when the wort is pitched with the proper amount of a pure brewer""s yeast culture (typically about 0.7-1.5 lb/bbl). After 24 hours, fermentation is established and proceeds at an accelerated rate. Fermentation typically proceeds for about 7 to 10 days. During this period, the wort temperature must be controlled, since the fermentation process causes the temperature of the wort to rise. Once the yeast has metabolized all the fermentable ingredients in the wort, it settles to the bottom and is subsequently recovered and recycled for use in pitching other brews. As the fermentation process comes to a conclusion, the temperature of the wort begins to drop. The fermented wort (termed xe2x80x9cgreen beerxe2x80x9d) is drawn off for storage in a cold room tank, or xe2x80x9cruh,xe2x80x9d where, its temperature is lowered to about 0-5xc2x0 C.
Processing and Packaging. The xe2x80x9cruhxe2x80x9d beer may be allowed to remain in the ruh tank for completion of the maturation process, or it may be transferred into a separate maturation tank upon further settling of any remaining yeast and other solids. Depending on the particular brewery, the beer is allowed to age from about 14 days to about 3 months. During this period, the beer clarifies and its flavor develops. Upon maturation, the beer generally is filtered to remove the yeasts and other solids.
The beer can undergo a single- or a double-pass filtration process. The double-pass filtration consists of two steps: a primary (coarse) filtration, and a secondary (fine) filtration. Filtered beer is subsequently stored in a finishing tank.
To prepare the beer for consumption, it is carbonated to a specified level. Then, depending on the form of packaging, the beer may be pasteurized. (In the case of the cold-filtered xe2x80x9cdraftxe2x80x9d beers, a microfiltration system is used to remove contaminants, thereby obviating the pasteurization step.) Beer packaged in cans and bottles is usually pasteurized, while beer packaged in kegs (and sometimes bottles) remains unpasteurized. After final processing of the packaged product (e.g. labeling, etc.), the beer is ready for shipment to the consumer.
Other conventional processing steps well known to those skilled in the art may be used instead of, or in addition to, the above-disclosed general brewing methods. For example, the fermented wort can be diluted with water to produce a low calorie (40 or fewer calories per 12 ounces), non-alcoholic malt beverage (less than 0.5 volume percent alcohol) that closely simulates conventional beer flavor, taste and mouthfeel.
The Attributes of Fermented Malt Beverages
Malt beverages, especially beer, possess attributes readily discernable by the consumer. These attributes include foam, flavor and clarity. Of these, flavor is ultimately the most important characteristic to the consumer.
Flavor (purity) and after-taste (refreshing feeling) are typically measured within the industry as having one of the following five grades:
1: Taste is not very clean and after-taste has no refreshing feeling.
2: Taste is not clean and after-taste has almost no refreshing feeling.
3: Usual.
4: Taste is clean and after-taste has refreshing feeling.
5: Taste is very clean and after-taste has very refreshing feeling.
Flavor stability is typically evaluated in the stored packaged product (usually at a storage temperature of 28xc2x0 C. for 15 days) as having one of the following five grades:
1: Significantly staled.
2: Staled.
3: Usual.
4: Fresh.
5: Very fresh.
In addition, an increasing number of consumers desire an all-natural beer product which demonstrates the above qualifies yet is entirely free of artificial additives or supplements.
It is known in the art that the malted barley may be replaced in whole or in part by a so called xe2x80x9cbrewing adjunct.xe2x80x9d Suitable brewing adjuncts include maize, rice, sugar and various syrups. A brewing adjunct used in the production of a wort, such as maize, is usually crushed and a mash formed separately from the malt mash by adding enzymes. Prehydrolyzed products can be mixed with the malt mash, and syrups can be added to the wort at the time the wort is boiled as described above. The use of brewing adjuncts needs to be carefully controlled in order to produce beer of acceptable taste and color. The use of adjuncts made from maize, rice and other grains expands the brewing ingredients beyond the traditional ones listed above. Such an approach is, however, not possible in certain countriesxe2x80x94in Germany, for example, the Beer Purity Laws enacted in 1516 (the xe2x80x9cReinheitsgebxc3x6txe2x80x9d) which limit brewing ingredients to barley malt, water, hops and yeast, are still followed.
Compounds added to the wort mixture prior to the primary fermentation step are termed xe2x80x9cprocessing aids.xe2x80x9d On the other hand, compounds added to the wort mixture after the primary fermentation step are termed xe2x80x9cadditives.xe2x80x9d The difference between the two is significant because the use of additives is regulated, whereas the use of processing aids is not.
Flavor
As noted above, flavor is a key factor in the quality of a malt beverage such as beer. It is important that a beer retains its original, fresh flavor and character during distribution and storage. Thus, off-flavors are a great problem for beer manufacturers and distributors. The lightstruck flavor is a well-known off-flavor formed during the storage of bottled beer, as is the off-flavor caused by contamination with microorganisms. Other off-flavors that are produced during storage are expressed as papery, cardboard-like, oxidized, or in general, stale. At room temperature, the stale flavor in canned or bottled beer begins to develop shortly after packaging, and gradually and continuously increases to the extent that most American manufacturers of beer recall their product from the market if it is more than about 4 months from the packaging date. Although the oxygen in a bottle or can of beer is typically consumed by the beer within 24 hours of packaging, the noticeable presence of a stale flavor generally does not appear for several weeks.
In the past, the stale flavor of oxidized malt beverages, such as beer, generally has been attributed to the combined effects of oxidation, light and heat. Most investigators have focused on methods of reducing oxidation in the finished product. For example, the present practice of delaying the staling of beer includes maintaining a low level of air (or oxygen) in the packaged beer by minimizing free head space. Modern beer-filling machines are designed to achieve very low air levels in the packaged product. Typically, the bottle is evacuated before it is filled with beer, or the air in the evacuated bottle is replaced with carbon dioxide before filling, or overfoaming the bottle is utilized to displace the head space gases with beer foam. All of these practices can produce air levels of less than 0.5 ml. per 12 oz. bottle. But even these low levels of air still allow beer to oxidize in 2-3 months.
The off-flavors are made more obvious when the malt beverage has been stored at elevated temperature (thermal reactions). The negative influence of isohumulones and melanoidins on the oxidation of alcohols at elevated temperatures has been known for many years. See, e.g., Hashimoto, Rept. Res. Lab. Kirin Brewery Co. Ltd. 19:1 (1979). However, although beer is ideally stored at cold temperatures, maintaining a uniformly cool temperature is not always possible during transport. This is a particular problem in hot and humid countries where the temperature averages 28-38xc2x0 C., even more so in those countries where modern refrigeration is not always available. Therefore, there is clearly a need for a reliable method of stabilizing beer flavor, which does not rely upon specifically controlled environmental conditions after the packaged product has left the brewery.
The Maillard Reaction
More than eighty years ago, Louis Maillard first investigated the reaction of reducing sugars with the free amino groups of amino acids and proteins. This complex reaction, termed the Maillard reaction, or non-enzymatic browning, is responsible for the aroma and taste in cooked or preserved foods. Specifically, it is know to be involved in the resulting color and aroma of fermented malt beverages, such as beer or sake.
As diagramed in FIG. 1, the Maillard reaction is initiated by the reaction of primary amines (from amino acids, proteins and nucleic acids) with sugars to form imines (Schiff bases) which undergo further rearrangement to form the Amadori products, which are responsible for the browning and fluorescent process, which subsequently results in the formation of numerous advanced glycosylation end products. Broadly, the advanced glycosylation end products are termed xcex1-carbonyl intermediates, including, for example, 1-deoxydiketoses and 3-deoxyaldoketoses. When the reduced sugar is glucose, as in the brewing of malt, one of the a-carbonyl intermediates is 3-deoxyglucosone.
Hundreds of compounds, including dextrins, polypeptides, alcohols, polyphenols, pyrrols, isohumulones, melanoids, fatty acids and aldehydes, as well as related precursors and intermediates, are involved during the brewing process in the Maillard reaction. For example, there are over 140 reductases and dehydrogenases in the superfamily of reductases involved in the Maillard reaction.
A wide range of carbonyl compounds are known to be reduced via the Maillard reaction during fermentation, particularly from malt and wort, and to produce off-flavors (see Meilgaard et al., Tech. Q. Master Brew. Assoc. Am. 12:151-168 (1975)). Two biological pathways control the level of carbonyl compounds in the final productxe2x80x94the formation of aldehydes from the oxyacid pools and the enzymatic removal of wort carbonyls by the brewer""s yeast.
Higher alcohols and the corresponding aldehydes are formed partly by anabolic processes from the main carbon source and partly through the catabolic pathway from exogenous amino acids. In addition, aldehydes produced during fermentation, mashing and boiling are known to be potential substrates for aldehyde dehydrogenases or reductases. Peppard et al, J. Inst. Brew. 87:386-390 (1981). However, recent studies have indicated that aldehyde-reducing systems are be more complex than previously assumed. See Collins et al., Proc. Congr. Eur. Brew. Conv. 23:409-416 (1991); Kronlof et al., Proc. Cong. Eur. Brew. Conv. 22:355-362 (1989). It is now recognized that many enzymatic systems are involved in the reduction of the carbonyl compounds into higher alcohols, and that each system probably operates with varying activities during the course of the fermentation process (Debourg et al., J. Am. Soc. Brew. Chem. 52(3):100-106 (1994). For example, carbonyl compounds, particularly unsaturated carbonyls, are not stable. Such compounds are decomposed to shorter chains, which are subject to aldol condensation.
Unsaturated aldehydes, notably trans-2-nonenal, and related compounds involved in the oxidation of long-chain fatty acids have long been associated with stale flavor in beer. See, e.g., Debourg et al., supra, and U.S. Pat. No. 4,110,480. It is well known that enzyme mediated oxidation of unsaturated fatty acids, such as linoleic acid, followed by the subsequent oxidative or non-oxidative scission of the carbon chain, will produce flavor-active compounds having carbon lengths of 6 to 12. Therefore, those attempting to stabilize fermented malt beverage flavor have, in some cases, focused on modifying the lipids involved in the brewing process. However, in beer, the lipids are derived from malt in various forms including simple lipids (fatty acids, triglycerides and other neutral lipids), complex lipids (glycolipids and phospholipids) and bound lipids such as those bound with starch grains.
Numerous methods have been attempted to remove lipids from the raw materials, including (1) removal of the germ of the grain, which contains a significant portion of lipids found in the raw material cereals (polishing), (2) removal of lipids from the raw material cereals by ethanol extraction, (3) pretreatment of the raw material cereal grains with a lipid decomposing enzyme (Japanese Patent Examined Publ. No. 22478/1973, Japanese Patent Unexamined Publ. No. 55069/1987), and (4) removal of lipids by special filtration-separation (U.S. Pat. No. 5,460,836). However, not all lipids have an adverse effect, i.e., the balance of these forms of lipids subtly affects the beer quality and the efficiency of beer brewing process. Thus, even after years of study, it remains unknown what balance is appropriate, or how altering the total lipid content will affect the stability of flavor in the stored, finished product.
Another recognized technique for stabilizing beer against oxidation is to add an oxygen scavenger, such as sulfur dioxide, usually in the form of bisulfite, to the beer. Sulfur dioxide is produced by yeast during fermentation and will combine with carbonyls to form bisulfite addition components that are hydrophilic, and thus less volatile. However, although effective, increasing the concentration of SO2, naturally or artificially, may be commercially unacceptable. In the United States, for example, SO2 is limited by law to less than 10 ppm, and even those low levels produce undesirable and sulfury aromas in some beers. In other countries, such as Germany, any addition of exogenous SO2 is strictly prohibited.
Even if permitted, the addition of bisulfite, which works by binding to aldehydes, is not an ideal solution. Beer is a complex product, comprising many different aldehydes (notably acetaldehyde, a normal by-product of fermentation), hence the action of a sulfite additive is often muted. The addition of other oxygen scavengers has also been tried, but with little effect on the long-term stability of the flavor in the fermented malt beverage.
In spite of all of the available art and years of research, however, beer flavor still goes stale. Thus, it is clear that until the present invention, there remained a long-felt need in the art for a reliable method of stabilizing the flavor of fermented malt beverages, which has the following characteristics: (1) will not significantly alter the desirable fresh flavor of the finished product, (2) will not significantly diminish the efficiency of the brewing process, (3) violates no law or regulation regarding the addition of additives or preservatives, and (4) is not dependent on maintaining specific environmental conditions for the transportation and storage of the packaged product.
The present inventors, deducing that the products formed during the Maillard reaction could be used as indices of beer aging, developed a method (using indices measured by a combination of capillary electrophoresis and HPLC techniques) to reliably monitor flavor stability and the organoleptic effect of aging on beer (Bravo et al., IBTC Technical Consortium Meeting #35, Salzburg, Austria, September 1993; Bravo et al., IBTC Technical Consortium Meeting #36, Caracas, Venezuela, November 1994). By utilizing the method for the detection of the relevant chemical indices, a novel system was developed, significantly advanced over those described and used heretofore, for dependably and efficiently assessing the degree of beer freshness, and for determining the storage conditions (time and temperature) of a beer exposed to a previously unknown environment. Furthermore, these analytical systems have been utilized to develop methods for improving the flavor stability of malt beverages such as beer, and for producing malt beverages by these methods.
In initial investigations designed to solve the above-described problems, it was discovered that by enzymatically regulating the production of certain intermediates of the Maillard reaction formed during the brewing process, a fermented malt beverage could be reliably produced having a refreshingly clean taste and enhanced flavor stability. The present inventors made further investigations based on this finding, and developed the present invention.
The present inventors have developed an entirely new method for stabilizing fermented malt flavor by focusing on an aspect of the brewing reaction not previously considered in the prior art. The present invention is therefore directed to the stabilization of the flavor of a fermented malt beverage using one or more inhibitors, blockers, reducing agents or binding agents that inactivate Maillard reaction intermediates; such agents may include, for example, NADPH-dependent oxidoreductase enzymes or chemical agents such as aminoguanidine.
In order to evaluate flavor stability, the inventors found it essential to have a sensitive, quick and reproducible method by which changes in the flavor of the beer could be analyzed. Sensory testing has been the traditional means available for assessing the organoleptic quality of beer. Taste testing, although sensitive, suffers from human limitations, such as personal bias and the tendency to make comparative (subjective) rather than objective evaluations (Mathews et al., Trends in Food Science and Technol. 4:89-91 (1990)). The Institute of Brewing Technology began using high performance liquid chromatography (HPLC) analyses according to e.g., Greenhoff and Wheeler, J. Inst. Brew 86:35 (1981); Strating and Drost, Dev. in Food Sci. 17:109-121 (1988). Improved methods utilizing purge and trap techniques, gas chromatography, and mass selective detection using the SIM technique were applied to establish higher capacity and better separation, determination and identification. See, e.g., Narzixcex2et al., MBAA Tech. Q. 30:48-53 (1993). However, objective measurements of a particular quality parameter are meaningless unless they are correlated to the human response to the beverage as a whole when it is purchased and consumed under normal conditions.
Thus, the present inventors developed a system by which the organoleptic deterioration of beer could be evaluated by analytical indices providing a series of compounds (see FIG. 2) representing a reproducible continuum of fresh through deteriorated (stale) forms. These analytical indices were then related to organoleptic evaluations, as demonstrated in FIGS. 3A and 3B, to provide a correlation between objective and organoleptic measures of flavor freshness. Bravo et al., IBTC Technical Consortium Meeting #35, Salzburg, Austria, September 1993; Bravo et al., IBTC Technical Consortium Meeting #36, Caracas, Venezuela, November 1994. These compounds participate in the reactions involved in the beer staling process (substrates, intermediates or final products), but do not necessarily produce the stale flavor. These analytical indices are relatively easy to detect and show a significant change in their relative peak areas during the aging process (see FIGS. 3A and 3B).
The concentration of furfural, 5-methylfurfuryl, 2-acetylfuran and 5-hydroxymethylfurfural (5-HMF) are useful indices for measuring heat damage in beer. For example, in an effort to establish a xe2x80x9cquality deterioration test,xe2x80x9d methods have been developed for detecting furfural and 5-methylfurfuryl in fruit juices during storage. Harayama et al., Agric. Biol. Chem. 55:393-398 (1991) found by multivariate analysis of off-flavor in head-space volatiles formed during the storage of beer, that certain furfural compounds were a valuable index for measuring a particular cardboard flavor in the beer. Grongvist et al., EBC Cong. 421-428 (1993), using gas chromatography to measure carbonyl compounds present during beer production and aging, found that the concentration of furfural was significantly increased during aging.
The present invention is directed to the production of malt beverages having improved flavor stability. The invention has particular utility in the production of fermented malt beverages such as beer, although the invention also may be advantageously used in the production of other malt flavored beverages. The invention is further directed to brewing methods for producing fermented malt beverages, such as beer, the beverages prepared by said method, and beverages having a substantially stabilized flavor.
In particular, the present invention is directed to a method for stabilizing the flavor of a fermented malt beverage, most particularly a beer, by the addition of one or more reductase enzymes including, but not limited to, oxidoreductases such as aldehyde reductases (EC 1.1, including aldose reductases, aldocarbonyl reductases and oxoaldehyde reductases), keto reductases (EC 1.2, including ketose reductases and ketocarbonyl reductases), acetyl reductases (EC 1.3), primary aminoreductases (EC 1.4), secondary aminoreductases (EC 1.5) and particularly NADH/NADPH oxidoreductases (EC 1.6, most particularly isozymes of Old Yellow Enzyme (OYE; EC 1.6.99.1) such as OYE1 (SEQ ID NO:1), OYE2 (SEQ ID NO:2) and OYE3 (SEQ ID NO:3)). The invention also relates to fermented malt beverages (particularly beers) prepared by these methods, and to fermented malt beverages (particularly beers) having enhanced flavor stability.
The invention further relates to the use during the brewing process of reductase enzymes such as those described above from naturally occurring sources (e.g., yeast cells such as Saccharomyces spp. cells and particularly Saccharomyces cerevisiae and Saccharomyces carlsbergensis cells), to stabilize the flavor of the resulting fermented malt beverage and to produce a fermented malt beverage having a stable flavor. It also relates to microorganisms, particularly yeasts, bacterial cells and animal cells (including insect cells) which have been specifically modified, selected, or genetically engineered to express or secrete one or more of the above-described reductase enzymes which may be used during the brewing process to stabilize the flavor of the resulting fermented malt beverage and to produce a fermented malt beverage having a stable flavor.
The present invention also provides enzymatic digests from naturally occurring sources (e.g., yeast cells) or from genetically modified cells (e.g., the genetically modified yeast, bacterial or animal cells described above), or extracts thereof, which will provide a sufficient amount of the necessary enzymes to block, inhibit or reduce the Maillard reaction intermediates (e.g., 3-deoxyglucosone), which results in the formation of the stale flavor in fermented malt beverages.
The present invention also provides methods for enhancing the flavor stability of a malt beverage. In accordance with the present invention, these methods are suitable for enhancing the flavor stability of a fermented malt-beverage, in particular a beer. Thus, it is an object of the present invention to provide methods of brewing or preparing beer, wherein the flavor stability of the beer is enhanced. A first such method of the invention comprises adding one or more of the above-described natural sources (e.g., yeast cells), genetically modified sources (e.g., genetically modified yeast, bacterial or animal cells), enzymatic digests or extracts, or purified reductase enzymes, and one or more reductase enzyme cofactors (such as NADH or NADPH) to the grain malt, wort mixture (prior to or following fermentation) or fermented malt beverage (prior to or following processing), under conditions that favor the enhancement of flavor stability in the finished fermented malt beverage. A second such method of the invention comprises immobilizing the above-described enzyme sources, digests or extracts, or purified enzymes, and reductase enzyme cofactors, on a solid support and contacting the grain malt, wort mixture (prior to or following fermentation) or fermented malt beverage (prior to or following processing) with these immobilized reductase enzymes/cofactors under conditions that favor the enhancement of flavor stability in the finished fermented malt beverage. According to this aspect of the invention, the solid support may be a membrane (such as nitrocellulose, diazocellulose, nylon, etc.), a bead (such as an alginate bead, a polystyrene bead, a latex bead, a glass bead, a magnetic or paramagnetic bead, etc.), a polystyrene plate, and the like. Most preferred are membranes and beads. In a particularly preferred embodiment of this aspect of the invention, one or more enzyme cofactors such as NADH or NADPH, and one or more isozymes of the NADPH oxidoreductase OYE (EC 1.6.99.1) such as OYE1 (SEQ ID NO:1), OYE2 (SEQ ID NO:2) or OYE3 (SEQ ID NO:3) or cells (natural or genetically modified) or extracts there of comprising one or more OYE isozymes, are immobilized onto a solid support and used in the methods of the invention to produce a fermented malt beverage, particularly a beer, having enhanced flavor stability.
The invention further provides the malt beverages produced by these methods. In accordance with the present invention, the malt beverage may a fermented malt beverage, particularly a beer. Thus, it is an object of the present invention to provide a beer in which the flavor stability has been enhanced.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.